Automated cellular analyzers, such as hematology analyzers, enable the counting and characterization of cells in a cellular sample. Certain of these instruments detect changes in electrical conductance as cells are drawn through a small aperture. Cells conduct electricity differently than their surrounding fluid. Therefore, their passage through the aperture alters its effective cross-section and hence its conductivity. The accurate counting and characterization of cells by these devices requires the ability to precisely measure the volume of sample analyzed during a count episode. One way to do this is to utilize liquid volumetric metering, where the flow of a specific volume of a fluid through a capillary controls the start and stop of a counting episode.
The metering system of automated cellular analyzers that use liquid volumetric metering can comprise a metering tube, a start sensor, and an end sensor. The start sensor and the end sensor are located at opposite ends of the metering tube. Both sensors can be optical sensors, calibrated to detect a change in light intensity that occurs as the meniscus of the metering fluid passes. The flow of a liquid metering fluid through the metering tube, first past the start sensor and then past the end sensor, triggers the start and stop of a counting episode.
Presently, automated cellular analyzers that use liquid volume metering have the metering tube and sensors oriented vertically such that the start sensor is at the top of the metering tube and the end sensor is at the bottom of the metering tube. The metering fluid flows from the top down, first triggering the start sensor and then the end sensor. In this orientation, the problem of liquid “side flow” can occur. Due to the effects of gravity on the liquid metering fluid, some of the fluid can flow faster along a side of the inner wall of the metering tube, changing the angle of the meniscus such that it will not be detected by the start and stop sensors. In this case, metering fails. To combat this problem, metering fluids are designed with relatively high surface tensions that reduce the probability of side flow. However, even with a metering fluid with a relatively high surface tension, side flow can occur if cellular debris deposits on the metering tube.
Recently, automated cellular analyzers with liquid volumetric metering systems that eliminate the side flow problem have been designed. These instruments use a metering tube oriented vertically, but the metering fluid flows from the bottom up. The start sensor is located at the bottom of the metering tube and the end sensor is located at the top of the metering tube. Thus, the side flow phenomenon is completely eliminated as the effects of gravity on the metering fluid are no longer relevant. The development of these instruments necessitated the development of novel metering reagents, as existing reagents were not suitable for use in these new systems.
Automated cellular analyzers are often arranged such that on one side of the aperture is a sample bath which receives the cellular sample suspended in an isotonic diluent prior to analysis. On the other side of the aperture is a rinse reagent that acts to flush cells away from the aperture after they have been counted. The rinse reagent is in fluid communication with the sample bath, and as such, back flow can occur wherein some of the rinse reagent enters the sample bath and comes into contact with the cellular sample. This is especially so when the instrument is not in use. Traditionally, the isotonic diluent used to suspend the cellular sample is also used as the rinse reagent. Thus, the fluids on either side of the aperture are traditionally the same.
Many of the commercially available metering fluids and rinse reagents for use in automated cellular analyzers produce formaldehyde in amounts in excess of about 400 parts per million. Formaldehyde has been classified as a known human carcinogen by the WHO International Agency for Research on Cancer (IARC), and is also toxic and allergenic. Increasingly, regulatory agencies in states like California and Massachusetts have been restricting the amount of formaldehyde allowed in industrial and medical waste. According to these regulations, formaldehyde concentrations in waste equal to or less than 1 part-per-million is considered formaldehyde-free. Consequently, diluents, metering fluids, and rinse reagents for use in automated cellular analyzers that produce less than 1 part-per-million (ppm) of formaldehyde over the course of their shelf-life are highly desirable.
The present invention improves upon the metering fluids currently available by disclosing stable metering fluids optimized for use in automated cellular analyzers that use a bottom-up metering tube arrangement. The metering fluids of the present invention have a relatively low surface tension and detergent capabilities that aid in preventing the deposition of cellular debris on the inner surfaces of the instrument, simplifying the cleaning and maintenance of the cellular analyzer. The metering fluids of the present invention are also isotonic and non-hemolytic, and thus are suitable for use as a rinse reagent. In contrast to the diluents traditionally used as rinse reagents, the compositions of the present invention have detergent capabilities. Thus, when also used as the rinse reagent, the metering fluids of the present invention aid in preventing the deposition of cellular debris in and around the aperture.
The metering fluids of the invention can also be used to flush out the cellular analyzer in between count episodes to remove cellular debris on the inner surfaces of the instrument. The metering fluid compositions of the present invention preferably also have broad biocidal activity, are isotonic, non-hemolytic, and produce menisci with angles that properly trigger the start and end sensors, beginning and ending the counting interval. Advantageously, the metering fluid compositions of the present invention also produces less than about 1 part-per-million of formaldehyde over the course of their shelf lives.